Standarization of mouse and rat intestinal flora, and development of preservation and monitaring methods for the intestinal flora.

本项目的目的首先是为SPF级小鼠和大鼠的肠道菌群标准化提供生理功能正常的诺生菌小鼠和大鼠，进而开发标准化的小鼠和大鼠肠道菌群保存与监测系统。我们的研究结果如下：1.利用已建立的灵芝小鼠的制备方法，建立了生理功能正常的灵芝(GB)大鼠。从常规(CV)大鼠粪便中分离出3株乳杆菌(L.acidophilus，L.Murinus)和28株拟杆菌属(Bacteroidaceae)菌株，并用氯仿处理的CV大鼠粪便(仅存活梭状芽孢杆菌)接种无菌(GF)大鼠。结果：1.这些ex-GF大鼠无艰难梭菌感染，口服接种后可消除铜绿假单胞菌感染。在接种GB大鼠粪便悬液的GF小鼠中，保存了GB大鼠的菌群。只有通过口服GB大鼠粪便和鼠菌群小鼠，才有可能产生GB大鼠。将保存在GB小鼠肠道内的标准化小鼠菌群接种于4个不同品系的GF小鼠(CF#1、IQI、NC、BALB/c)。不同品系的GF小鼠在盲肠大小和粪便菌群组成上没有差异。在转GB大鼠建立的SPF大鼠群体中，其粪便菌群组成稳定。对标准化小鼠菌群冷冻保存方法进行了研究。最后，在1：100的营养肉汤中加入10%或20%甘油，在-80℃保存1年。将保存好的花悬液接种到GF小鼠体内，可获得生理功能正常的Gb小鼠，其花组成与原Gb小鼠相似，比在Gb小鼠肠道中保存1年的花组成更接近。

The purpose of this project is first of all to produce of gnotobiotic mice and rats with normal physiological function for standardizing the intestinal flora of SPF mice and rats, and then to develop the preservation and monitoring system of standardized intestinal flora of mice and rats. Our results were as follows :1. Gnotobiotic (GB) rats with normal physiological function were produced by using the methods which already have established for production of gnotobiotic mice. Namely, 3 strains of Lactobachillus (L.acidophilus, L.murinus, L.salivarius) and 28 strains of Bacteroidaceae isolated from feces of conventional (CV) rats, and chloroform-treated CV rat feces (survive only clostridia) were inoculated into germfree (GF) rats. These ex-Gf rats were free from Clostridium difficile and eliminated Pseudomonas aeruginosa inoculated orally.2. Gb rat flora was kept in GF mice inoculated with fecal suspension of GB rats. The production of GB rats was possible only by oral inoculation of feces of GB rats and rat-flora-mice.3. Standardized mouse flora kept in the intestine of GB mice were inoculated into 4 different strains of GF mice (CF#1, IQI,NC,BALB/c). There were no differences in cecal size and the composition of fecal flora among GF mouse strains.4. In SPF rat colony established by transferring GB rats, composition of their fecal flora was kept stably in the colony.5. Preservation method for standardized mouse flora by freezing was examined. Finally, in 1 : 100 fecal suspension in nutrient broth with 10 or 20% glycerol kept in -80 C floral bacteria were kept for 1 year. By inoculation with preserved floral suspension into GF mice, GB mice with normal physiological function were able to be produced, and their floral composition was similar to original GB mice more than that kept in GB mouse intestine for 1 year.